KR810000257B1 - Method of extracting antioncogenic nitrogen-containing polysaccharides - Google Patents

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Description

Extraction Method of Nitrogen-Containing Polysaccharides with Anticancer Activity

The present invention relates to a method for extracting nitrogen-containing polysaccharides having anticancer activity.

The present invention relates to a method for obtaining anticancer substances in high yield by extracting fungi belonging to basidiomycete (Basidiomgcetes steel) with a water-soluble solvent under certain conditions.

It is well known that anti-cancer substances, which are polysaccharides, can be prepared by purifying extracts obtained by extracting fungi belonging to basidiomycetes with water-soluble solvents under normal pressure. However, this known manufacturing method is very poor because the extraction effect of the active ingredient is very bad as the extraction method applied here.

This low extraction efficiency is due to the large loss of active ingredients due to the large amount of active ingredients remaining in the extraction residue. This is not very desirable, not only for the production of antibiotics, but also for industrial reasons, as these losses both result in the disposal of valuable materials and the disposal of waste materials that must be discarded.

While studying the method for solving the above-mentioned problem in the extraction method of anticancer substance obtained by extracting fungi belonging to basidiomycete with water-soluble solvent, basidiomycetes (Bassidio mycetes) under the temperature and pressure of 120-200 ℃ When the fungi belonging to the steel) were extracted with a water-soluble medium, it was found to be a polysaccharide containing nitrogen and have a very good anticancer activity.

As used herein, the fungus belonging to the fungus fungi means fungi with fungi and / or spores, and the fungus fungi of the present invention include polyporaceae, and chromonophoracea in plants. (Mucronoporaceae), Trichoromataaceae, Hygrophoraceae, Volttiaaceae, Strophariaceae, and Cortinarriaceae Rhodophllaceae family, Boletaceae family, Hydnaceae family, Fistulinaceae family Tremellaceae family, Parinea family Phalaineae, clathraceae, Cravariaceae, Corticiaceae, Agaricicaceae, Coprinaceae , Rususulaceae, and Aurikurariase Auriculariaceae, coniophoraceae, and Lycoperdalaceae, Rhizopogong and Calostoma.

These basidiomycetes are known and include, for example: trametes sanguinea, Trametes ciunaabaria, Polyporellus sqaumosus (Fr. ) Karst), Daedaleopsis styracina (P. Henn. Et shir) lmazeki, Fomitopsis pinicola (Fr.) Karst, Lenzites butulina (Fr) .., Groeophyllum saepiarium (Fr. Karst), Ganoderma boninense pat., Fomes fomentavius (Fr.) kickx, Prephora Grifola gigantea (Fr.) Pilat, Favorus alveolarius (fr.) Qu'l, Coriolus versicolor (Fr.) Que'l, Coriorus Coriolus hirsutus (Fr.) Quel, Coriolus pargamenus (Fr.) Pat., Corio Coriolus consors (Besh.) Imaz, Coriolus conchifer (schw.) Pat, Coriolus pnbescens (Fr.) Quel, Coriolus Viformis (Coriolus biformis (klotz), Hiroschpoorus abietinus, Laetiporus sulphureus, Grifola frondosa, Porodidisus penmurus, Rigidoporus ulmarius, Oxyporus ravidus, Tyromyces lacteus, Cryptoporus volvatus, Poria vaporaria, Irpex lacteus, Lampteromyces japonicus, Tricholomopsis rutilans (Fr.) sing, Tricholoma rutilans (Fr.) Quel , Armillariellamered lled), Lacaria amethystina, Ischnoderma (Fr.) Karst, Polyporus resinosum (Fr.), Lyophyllum ulmarium ), Lyophyllum aggregatum, Trichoromama matsutake, Hohenbuhelia serotina (Fr.) sing., Cantharellula cyathiformis (Fr. Sing, Pannellus stipieus (Fr. Karst), Schizophyllum commune (Fr.), Panus conchatus (Fr.), Freu Pleurotus ostreatus (Fr.Que, l), Oudemausiella radicata (Fr.) Sing, Lentinus edodes (Berk.) Sing, Prammurina Beruru Flammulina velutipes, Mycena pura, Lepista subnuda, Crititobe Infundiven Lipour Clitocybe infundibnliformus, Tricholomophis platyphylla (Fr.), Collybia platyphylla (Fr.) kummer, Tremelodon gelatinosum (Fr. .,)), Areurodiscus amorphus Rabenh, Lycoperdon pyriforme Pers., Phaeolus schweinitzii (Fr.) Pat, Cryptoder mafinini (Cryptodeder mapini (Fr.) Imazeki), Pelinus gilvus (Fr.) Pat, Stereum hirsutum Fr., Merulusi tremellosus Fr., Roparian Mira Virism (Lopharia mirabilis Berk. etBr. Pat), Echinodontium tsugecola (P. Henn. et shirai), Hygrocybe ovina (Fr.) Kuhn, Hygrophorus ovinus Fr. ), Agaricus bisporus (lange) sing, Psalliota hortenisis (Cke.) WG Smithvar.bispora Lange, Phaeolepiota (Fr.) Maire, Poriota Aurea (Pholiota aures (Fr.) Gill); Cystodermaaureum (Fr. kuhn.et Romgn.); Pholiota vahlii (Fr.), Lange, Coprinus aframenta Coprinus atramentarius (Fr.) Fr, Agrocybe arvalis (Fr.) Fayod var.tuberigena (Quel), Konr. Et Maubl, Naucoria arvalis (Fr.) Quel Quel Var.thberigena Quel, Agrocybe tuberosa (Henn) Sing, Qymnoplius aeruginosus (Peck) Sing., Poly Pholiota aeruginosa (Peck), Rhodophyllus murraii (Berk. Et Curt.) Sing, Entoloma murraii (Berk. Et Curt. Sacc.), Whale Boletus deulis Fr., Lactarius volemus Fr., Auricularia polytricha (Mont.) Sacc., Rozites caperata (Fr.) Karst., Pholiota caperate (Fr.) Gill, Suillus granulatus (Fr.) Knntze, Betus granulatus (Fr.), Rusura de Russula delica Fr., Russula Chloroides (Krombh Bers.), Phlogiotis helvelloides (Fr. Martin), and Guepinia helvelloides Fr. , Gyrocephalus rufus Bref., Gyroporus castaneus (Fr.) Quel, Aruricuraria Auricularia auriculajudae (Fr.) Quel, Cyrophana lacryman (Fr.) Pat, Merulius lacrymans Fr., Onnia orientalis (Lolyd) ) Imazeki), Polyporus orientalis Lloyd, Fuscoporia obiligua (Fr.) Aoshima, Poria obligua Fr., Inonotus cuticularis ( Fr.) Karst), Polyporus cuticularis Fr., Polyporus mikkadoi Lloyd, Hygrophorus leucophaeus (Fr.) Gill, Agrosibe Agrocybe cylindracea (Fr. Maire), Pholiota chlindracea (Fr.) Gill, vl aerita (P. aegelita (Fr.) Maire, Poliota cylindracea (Fr.) Gill, P. aegelita (Brig.) Quel, Naematoloma sublateritium (Fr.) Karst, Pholiota nameko (T. Ito) S. Ito et Imai, Pholiota glutinosa Kawam, Cortinaarius elatior Fr., Pholiota glutinosa Kawam, Cortinarius elatior Fr., Rhodophyllus abortivus (Berk. Et Curt Sing.), Crytopyrus avor Titopilus avortivus (Bork et curt.) Sacc., Suillus grevillei (klotzch) Sing., Creolophus spathus spathulatus Imazcki, Fistulina hepatica Fr.), Protodaedalea hispida Imazeki, Dictyophorai ndusiate (Pers.) Fisch, Lysurus mokusin (Pers.), Fr.f.sinensis (Lly \ oyd) Kobayasi, Lycoperdon gemmatum Fr., Rizopogon Lubesen (Rhizopogon rubescens (Tul.) Tul), Calostoma japonicu P. Henn., Ramaria flava (Fr. Qule), Clavaria flava Fr. , Rhodophyllus Rholopoli us (Fr.) Pudi, Entoloma Pholdpolium (Fr.) Quel, [Roguya Imajanki and Zhou Hongo, ＂Coloured Illustrations of Fungi of Japan's Volume I (1974) and II (1975).

Thus, the main object of the present invention relates to a method for extracting the mycelium and / or suicide of the aforementioned terminal fungus to extract a substance having excellent anticancer activity in a high yield.

The other aims of this expression are evident by considering the following in detail.

A feature of the present invention is that the genus belonging to basidiomycetes can be extracted with a water-soluble solvent at a temperature and pressure of 120 ° to 200 ° C. to obtain an anticancer substance which is a polysaccharide containing nitrogen rule.

In general, extracting basidiomycetes under high temperature conditions, such as 120 ° to 200 ° C, can decompose the active ingredient of mold and increase the overall extraction rate. .

In contrast to the general idea, however, the present invention reveals that high yields can be obtained if extracted under pressure under these temperature conditions.

In order to extract under pressure according to the present invention, the mycelium and / or fruiting body of the basidiomycete gompie, which is a starting material, are put together with a water-soluble solvent in a pressure vessel such as a batch type autoclave or a fluidized tubular extractor, and the substance therein is added to 120 to Extract at a temperature between 200 ° C.

The pressure applied to this treatment is about the same as the total vapor pressure of the water soluble solvent in the vessel.

It is usually not higher than 200 kg / cm 2 and preferably smaller than 16 kg / cm 2 but not lower than 1.8 kg / cm 2. The extraction time is appropriately adjusted depending on the applied temperature, but is usually within 5 to 360 minutes. The appropriate extraction time is chosen according to the temperature applied in the range of 120 ° C. sowl 200 ° C. within the above mentioned range.

If the extraction temperature is less than 120 ℃ satisfactory extraction effect is not obtained, while if the temperature exceeds 200 ℃ the active ingredient is decomposed and can not be a practical recipe. Also, if the export time is less than 5 minutes, the extraction effect is not good, while if the extraction time is longer than 360 minutes, it should be noted that the active ingredient may be degraded.

The extraction process in the practice of the present invention may be carried out repeatedly under the above-mentioned conditions, or the starting material (molecular fungus) may be immersed in an aqueous solvent at a temperature lower than 100 ° C. before the extraction process, or otherwise obtained The extract may be washed with an aqueous solvent at a temperature lower than 100 ° C. after extraction.

The water-soluble solvent used in the present invention may be used in common base methods such as pure water, aqueous solutions of alkaline solutions, urea solutions and amino acid solutions.

The amount of such an aqueous solution is usually used in a ratio of 5 to 100 times with respect to the starting material, it is preferable to use in a ratio of 10 to 50 times (weight ratio). The most preferred water-soluble solvent used in the present invention is water or a dilute alkaline solution of 1 N or less.

The extract thus obtained is then purified by ultrafiltration, salting out, dialysis, reversible osmosis or other methods alone or in combination to yield and purify low molecular weight substances up to 5000 in the extract. This purification method yields a polysaccharide containing nitrogen with excellent anticancer activity.

Nitrogen-containing polysaccharides obtained according to the method of the present invention has excellent anti-cancer activity with a particularly high inhibitory action against hard Sarcoma-180 cancers by oral administration as well as intraperitoneal administration in rats. will be.

This means that the polysaccharide containing nitrogen according to the present invention can be used as an excellent anticancer drug, and this effect is confirmed by various experiments.

The substance obtained according to the present invention is not only by oral administration to anticancer action, but also stimulates the immune response. More specifically, this substance has no side effects in chemotherapy for cancer patients and increases sensitivity to radiation therapy, as well as a decrease in the patient's immunity or physical ability due to surgery or blood transfusion or when the patient is immune. Or when they are weak in physical capacity, they make them resistant to viral or bacterial infections. In addition, oral administration of the substance according to the present invention has an excellent effect on enhancing liver function, improving appetite, treating disorders of the gastrointestinal tract, and improving diuresis. This is also valid for the treatment of leprosy.

As described above, according to the preparation of the present invention, in addition to the above-mentioned other effects by oral administration as well as intraperitoneal administration, nitrogen-containing polysaccharides having excellent anticancer activity in high yield are described in the following description of the present invention as follows. It can be obtained by the relatively simple preparation described.

Thus, the present invention makes a very good technical contribution in the industrial extraction of anticancer substances from the terminal fungus.

The present invention is now described in more detail by the following specific description.

Example 1

The mycelium of Coriorus Bersicoro (Fr) Quelle obtained by artificial culture was dried to 10% water, divided into 5 parts, each part was mixed with 12.5 times (weight ratio) water and put into the autoclave. Extraction is performed under extraction conditions as shown in Table 2. The extract obtained from each part was then filtered and the extract solution was fractionated, and the obtained extract solution was dialyzed with water refluxed using a diasing tube (Visking tube, manufactured by Union Carbide Co., Ltd.) at 5 ° C. for 72 hours according to a normal method. give.

The treatment by this dialysis method removes a substance having a low molecular weight (molecular weight 5,000 or less) in the extract. The dialysed solution is further concentrated to give a reddish brown powder with 7% moisture. This powder material has an average molecular weight of 10,000 or more, and elemental analysis and various color reaction tests as shown in Table 1 show that the material is nitrogen-containing polysaccharide.

TABLE 1

Color reaction test

From this test it will be understood that the products of the invention are primarily polysaccharides containing nitrogen in the peptide bound state. In order to confirm the anticancer effect of the nitrogen-containing polysaccharide obtained in the present invention, the following test is carried out.

[Test Methods]

Sarcoma-180 cancer cells are implanted into the rat abdominal cavity and after 7 days (after sufficient cell proliferation), 106 cells are transplanted again into the subcutaneous area of the armpit of another group of mice to develop a hard cancer. Intraperitoneal administration of a substance of the present invention begins 24 hours after transplantation. A single dose of 10 mg / kg, ie 0.2 ml 20 g (weight of rat), is administered 10 times, once a day, across the day. On the 25th day after transplantation, each rat's cancer is weighed for root removal.

The degree of cancer growth inhibitory activity of the substance of the present invention is calculated from the average value of the cancer weights of the surrounding groups to which the substance of the present invention is administered and the average of the control cancer weights.

The measurement results are summarized in Table 2.

The contents of the various formulations used in this test, ie the amount of water-soluble solvent (water) used, extraction temperature and extraction time, selectivity and yield are also shown in Table 2.

TABLE 2

Note: (1) For comparison, samples prepared using molds such as Specimen 1-5 according to the present invention or extracted at normal pressure according to a conventional method.

(2) "Extraction rate" is the dissolving rod of a starting fungus, expressed as a percentage by weight.

(3) "Selectivity" is the yield of nitrogen-containing polysaccharides having a molecular weight of 10,000 or more obtained from the extract.

(4) Yield for mold is the yield of nitrogen-containing polysaccharides with a molecular weight of 10,000 or more for all molds supplied.

In the test of mice subcutaneously implanted with Sarcoma-180 cancer cells, each of the nitrogen-containing polysaccharides obtained under the extraction conditions according to the present invention exhibits the same or higher anticancer activity as compared to similar preparations obtained under conventional extraction conditions. It will be appreciated from Table 2 that the yield is greatly increased for the mold used.

Example 2

The mycelium of the same basidiomycete used in Example 1 was divided into 8 groups, and each group was mixed with 12.5 times the alkali or substantially neutral solution shown in Table 3 below, and placed in an autoclave. Extract under the pressure and temperature conditions shown. After extraction, neutralize the material of the autoclave, filter, and separate the insoluble to extract residue to fractionate the extraction solution.

This extraction solution is subjected to dialysis according to the method of Example 1 to obtain a substance having a powder amount of 10,000 or more. When the material is subjected to various color reactions shown in Table 1, it is confirmed that it is a polysaccharide containing nitrogen.

In order to confirm the anticancer activity of the nitrogen-containing polysaccharides thus obtained, the substances of Samples 1 to 8 were administered to mice subcutaneously transplanted with Sarcoma-180.

The results show that these substances have the same or higher anticancer activity as compared to the polysaccharides containing nitrogen obtained by extraction with ordinary hot water under normal pressure.

TABLE 3

Note: (1) Comparison is a sample prepared by using the same fungus as Sample 1-8 of the present invention according to the extraction method by hot water at normal atmospheric pressure.

(2) Other matters are as mentioned above in Table 2.

Example 3

Distilled crude fruit of Lentinus Edodes with 88.27% of the commercially available water was divided into 6 parts, and each part was mixed with an amount of water as shown in Table 4 below and placed in an autoclave. Extract under.

After extraction, the treated material is filtered in an autoclave and the filtrate is fractionated from the remaining insoluble residue as extraction residue. The obtained filtrate is dialyzed by the method described in Example 1 to obtain a fraction having an average molecular weight of at least 10,000. This dialysis treatment removes low molecular weight substances from the extraction solution.

Each of the materials thus obtained is subjected to various color reactions and elemental analysis tests as shown in Table 1, and these test results confirm that these materials are polysaccharides containing nitrogen.

Each material thus obtained shows a high percentage of inhibition of at least 80% by the same method as described in Example 1.

The progress and extraction conditions of the extract obtained in this example are as follows:

TABLE 4

Note: Each item in this table is described in Table 2.

Claims (1)

A method for extracting nitrogen-containing polysaccharides having anticancer activity, characterized by extracting fungi belonging to basidiomycetes as a water-soluble solvent at a temperature ranging from 120 to 200 ° C. under a pressure ranging from 1.8 to 20 kg / cm 2.